This invention relates to a hybrid plasmid and, in particular, a hybrid plasmid useful for expressing foreign genes in B. subtilis. Considerable interest exists in the application of genetic engineering techniques for the production of commercially valuable products such as insulin, human and animal growth hormones and enzymes. Much of the work to date has involved use of Escherichia coli as the host into which foreign genetic material is introduced. Expression of the genetic material in E. coli results in production of desired products. When combined with growth of genetically engineered cells in culture, it permits production of the desired products in commercially meaningful yields. Unfortunately, use of E. coli as a host is associated with certain disadvantages. As a result, alternative hosts, including other bacteria and yeast, are under investigation.
One particularly promising host for commercial applications of genetic engineering is Bacillus subtilis. B. subtilis is a nonpathogenic, gram-positive bacterium which is eaten daily by millions of Japanese as part of a fermented soybean product. B. subtilis may be the safest bacterium in which to achieve expression of foreign genes whose products, e.g. interferon, will be purified and subsequently injected into humans for at least two reasons. First, B. subtilis is known to be nonpathogenic. Secondly, E. coli is known to produce endotoxins which may contaminate genetic products and induce endotoxic shock in humans.
Bacillus subtilis is a prokaryote that has been used for gene cloning. Phenotypic expression of foreign genes in B. subtilis has been until recently obtained only with genes originating in gram-positive species such as Bacillus staphylococcus and Streptococcus, see for example Lovett et al., J. Bacteriol. 127, 817-828 (1976); Ehrlich, Proc. Natl. Acad. Sci. 74, 1680-1682 (1977); Keggins et al., Proc. Natl. Acad. Sci. 75, 1423-1427 (1978); and Gryczan et al., Proc. Natl. Acad. Sci. 75, 1428-1432 (1978). Goldfarb et al., Nature 293, 309-311, (1981) disclose the expression in B. subtilis of E. coli chloramphenicol resistance by supplanting the native regulatory element(s) of the gene coding for this activity with B. subtilis DNA fragments.
European Patent Application No. 81300858.8, Publication No. 0,036,259, discloses a method and a cloning vector for the controlled accumulation of cloned heterologous gene products in Bacillus subtilis. The cloning vector is capable of being replicated in B. subtilis and includes the heterologous gene located and oriented such as to be under the control of an operator, promoter and ribosomal binding site sequence. The gene codes for a protein which is under the control of a transport mechanism by which the protein is secreted by the B. subtilis.
European Patent Application No. 82302027.6, published on Oct. 27, 1982 as Publication No. 0,063,494, discloses a method and cloning vectors useful for the production of cloned heterologous gene products in B. subtilis. Use of the method and vectors allows the host to produce the heterologous gene product as a single unfused peptide having no extraneous amino acids attached that will accumulate within transformed host organisms. The publication specifically discloses the use of a beta-lactamase promoter to express human fibroblast (.beta.) interferon in B. subtilis.
Williams et al., Gene 16, 199-206 (1981) disclose a plasmid pPL608 which can be used to express the mouse dihydrofolate reductase (DHFR) gene and a segment of the E. coli trp operon in B. subtilis. The cloned mouse gene confers trimethoprim resistance on B. subtilis. The mouse gene was inserted at a PstI site preceding a chloroamphenicol acetyltransferase (CAT) gene present on pPL608 and its expression is not chloramphenicol inducible. The pPL608 plasmid has a mass of about 3.3 Md and consists of a major portion of pUB110 joining to a 0.8 Md segment of B. pumilus DNA containing a CAT gene plus a 0.2 Md EcoRI* promoter fragment cloned from phage SPO2 DNA.
Copending U.S. patent application Ser. No. 307,604, filed Oct. 1, 1981 (now abandoned) and designating P. S. Lovett as inventor, discloses a plasmid useful for introducing into B. subtilis foreign DNA, the nucleic acid sequence of which codes for the production of a desired product, comprising a double-stranded DNA molecule which includes a promoter DNA sequence which is not derived from B. subtilis plasmid DNA and a DNA sequence derived from a B. subtilis plasmid. The application discloses that the foreign DNA can include a gene coding for the production of a polypeptide product such as insulin, .alpha.-thymosin, growth hormones, enzymes, antibodies and the various interferons. The application further discloses that preferably the promoter is obtained from SPO2 and the B. subtilis plasmid source is B. subtilis plasmid pUB110.